There is a current need to find alternate energy sources to substitute for the use of fossil fuels for transportation applications. Biologically produced ethanol has been proposed as an alternative to petroleum-derived liquid fuels. There are different ways to generate ethanol through biological means. Obtaining ethanol from grains and seeds has been criticized for contributing to rising food prices and leading to deforestation. The disadvantages of obtaining ethanol from grain and cellulosic sources are further explained in paragraph [0007] of published U.S. patent application 20090017512 [H. D. May, T. Shimotori]. The present invention is addressed to the direct production of ethanol from carbon dioxide and water using genetically-modified cyanobacteria and overcomes problems associated with grain or cellulosic sources of ethanol.
Further, the present invention discloses the capability to produce ethanol using desert lands and salt water and resolves problems associated with demand on food-producing land and water resources. Moreover, the ethanol productivity of the present invention is higher than for corn-based ethanol. The present invention has projected productivity of 6,000 gallons (22,700 liter) ethanol per acre compared to 370 gallons (1,400 liter) ethanol for corn ethanol. [Bryan Walsh, “Biofuels: the New Alchemy,” TIME magazine, http://www.time.com/time/specials/packages/aricle/0,28804,18721 10—1872133—1872143-1,00.html; see also Emily Waltz, “Biotech's Green Gold,” 27 Nature Biotechnology 15-18 (2009)].
The present invention improves upon work disclosed by Woods et al. in U.S. Pat. Nos. 6,306,639 and 6,699,696, which taught the genetic modification of Cyanobacteria by incorporating the genetic information encoding for pyruvate decarboxylase (pdc) and alcohol dehydrogenase (adh). Specifically, the coding sequences of pyruvate decarboxylase (pdc) and alcohol dehydrogenase II (adh) from the bacterium Zymomonas mobilis were cloned into the shuttle vector pCB4 and then used to transform the cyanobacterium Synechococcus sp. strain PCC 7942. The pdc and adh genes were expressed at high levels, and the transformed cyanobacterium synthesized ethanol, which diffused from the cells into the culture medium. See also Deng and Coleman, Applied and Environmental Microbiology, February 1999, p. 523-528, Vol. 65, No. 2.
Methods to improve ethanol production in such ethanologenic organisms are needed to facilitate commercial implementation of this ethanol source. The ability to modify the genetics of specific species has been stated to be currently limiting progress. E. T. Johnson and C. Schmidt-Dannert, “Light Energy Conversion in Engineered Microorganisms, Trends in Biotechnology, Volume 26, Issue 12, December 2008, Pages 682-689. The problem of genetic engineering is complicated. Some of the obstacles to achieving high yields of products are a result of the interdependence of metabolic networks, which are strongly influenced by the global levels of a handful of metabolites: ATP/ADP, NAD+/NADH, NADP+/NADPH, and acyl-CoAs. ( . . . ) The incorporation of new pathways for biofuel synthesis can destabilize the balance of these important metabolites, leading to the production of undesirable byproducts and a decrease in yield. Sung Kuk Lee, Howard Chou, Timothy S Ham, Taek Soon Lee, Jay D Keasling, Current Opinion in Biotechnology, Volume 19, Issue 6, December 2008, Pages 556-563. Generally, on biochemistry, see Biochemistry. Fifth Edition. Berg J M, Tymoczko J L, and Stryer L. New York. W.H. Freeman and Company. 2002.
One way to increase ethanol production in a microbial host cell is to optimize the activity of the adh enzyme. The initial work of Woods et al. disclosed the use of alcohol dehydrogenase II (adh) from the bacterium Zymomonas mobilis. The present invention discloses how different selections of adh can be successfully made to increase ethanol production over that disclosed in the art.